Multiplexed luciferase reporter assay systems

Abstract

Inhibitors of luciferase enzymes are disclosed and find use in multiplexed assays using multiple luciferases and multiple inhibitors, in both in vitro and in vivo embodiments.

What is claimed is: 
     
       1. A method of detecting a plurality of distinct luciferase molecules comprising:
 (a) providing a plurality of distinct luciferase molecules in combination with substrates therefor, wherein said plurality of distinct luciferase molecules includes at least  renilla  luciferase and firefly luciferase; 
 (b) providing a plurality of distinct luciferase inhibitors, at least one of which selectively inhibits  renilla  luciferase and one of which selectively inhibits firefly luciferase molecules; 
 (c) sequentially measuring chemiluminescence from said plurality of distinct luciferase molecules prior to and after combination of said plurality of distinct luciferase inhibitors with said plurality of distinct luciferase molecules, 
 whereby the addition of said  renilla  luciferase inhibitor permits the detection of chemiluminescence from non- renilla  luciferase molecules, and the addition of said firefly luciferase inhibitor permits the detection of chemiluminescence from non-firefly luciferase molecules. 
 
     
     
       2. The method of  claim 1 , wherein said plurality of distinct luciferase inhibitors are provided sequentially. 
     
     
       3. The method of  claim 1 , wherein said plurality of distinct luciferase inhibitors are provided simultaneously. 
     
     
       4. The method of  claim 1 , wherein said plurality of distinct luciferase molecules comprises 2, 3, 4, 5 or 6 distinct luciferase molecules, 
     
     
       5. The method of  claim 1 , wherein said plurality of distinct luciferase inhibitors comprises 2, 3, or 4 distinct luciferase inhibitors. 
     
     
       6. The method of  claim 1 , wherein said plurality of distinct luciferase molecules further comprises one or more of gaussia luciferase,  cypridina  luciferase, gaussia luciferase lacking a secretion signal sequence or having an endoplasmic retention sequence, or  cypridina  luciferase lacking a secretion signal sequence or having an endoplasmic retention sequence. 
     
     
       7. The method of  claim 1 , wherein said plurality of luciferases are expressed from cells in culture. 
     
     
       8. The method of  claim 7 , further comprising separating said cells from cell medium. 
     
     
       9. The method of  claim 8 , further comprising disrupting said cells to form a cell lysate. 
     
     
       10. The method of  claim 1 , wherein said substrates comprise two or all three of D-luciferin, oxyluciferin, and coelenterazine. 
     
     
       11. The method of  claim 1 . wherein an inhibitor is selective or specific for  renilla  luciferase. 
     
     
       12. The method of  claim 11 , wherein said inhibitor selective or specific for  renilla  luciferase has the formula: 
       
         
           
           
               
               
           
         
         wherein a is substituted or unsubstituted alkyl (C 1 -C 10 ), substituted or unsubstituted alkenyl (C 1 -C 10 ), substituted or unsubstituted alkynyl (C 1 -C 10 ), substituted or unsubstituted alkyoxy (C 1 -C 10 ), amido or halide, b is H, substituted or unsubstituted alkyl (C 1 -C 10 ), substituted or unsubstituted alkyoxy (C 1 -C 10 ), substituted or unsubstituted alkenyl (C 1 -C 10 ), substituted or unsubstituted alkynyl (C 1 -C 10 ), amido or halide, and c is H, substituted or unsubstituted alkyl (C 1 -C 10 ), substituted or unsubstituted alkyoxy (C 1 -C 10 ), substituted or unsubstituted alkenyl (C 1 -C 10 ), substituted or unsubstituted alkynyl (C 1 -C 10 ), amido or halide. 
       
     
     
       13. The method of  claim 1 , wherein an inhibitor is cross-reactive with  renilla  luciferase and firefly luciferase. 
     
     
       14. The method of  claim 1 , wherein an inhibitor is selective or specific for firefly luciferase. 
     
     
       15. The method of  claim 14 , wherein said inhibitor selective or specific for firefly luciferase has the formula: 
       
         
           
           
               
               
           
         
         wherein a is H, substituted or unsubstituted alkyl (C 1 -C 10 ), substituted or unsubstituted alkyoxy (C 1 -C 10 ), substituted or unsubstituted alkenyl (C 1 -C 10 ), substituted or unsubstituted alkynyl (C 1 -C 10 ), amido or halide. 
       
     
     
       16. The method of  claim 1 , wherein said method comprises measuring firefly lueiferase,  renilla  luciferase, and one or more of gaussia luciferase,  cypridina  luciferase, gaussia luciferase lacking a secretion signal sequence or having an endoplasmic retention sequence, or  cypridina  luciferase lacking a secretion signal sequence or having an endoplasmic retention sequence. 
     
     
       17. The method of  claim 1 , wherein said method is peformed in an intact cell. 
     
     
       18. The method of  claim 1 , wherein said method is peformed in a cell-free system.